Cell and tissue transplantation is fast becoming an important treatment for several diseases and conditions including, but not limited to, diabetes (e.g., Janjic et al., Pancreas 13: 166-172, 1996), infertility (e.g., Warnes et al., Hum Reprod 12: 1525-1530, 1997), heart valve replacement (e.g., Feng et al., Eur J Cardiothorac Surg 6: 251-255, 1992), cataracts (e.g., Taylor Cryobiology 23: 323-353, 1986), skin replacement (e.g., De Luca et al., Burns 15: 303-309, 1989), and plastic and reconstruction surgery (e.g., Hibino et al., J Craniomaxillofac Surg 24: 346-351, 1996). Much of the advance in tissue transplantation is due to the discovery of bioartificial organs ("BAOs"). Generally, BAOs are comprised of cells which produce a desired biologically active molecule. The cells are encapsulated in a biocompatible permselective membrane or jacket to form a capsule. The pore size of the membrane is selected to permit diffusion of the biologically active molecule out of the capsule and to protect the encapsulated cells from the host's immunological system. These BAOs are well known in the art.
As cell transplantation in general, and BAOs in particular, gain wider acceptance and use, the need for long term storage of transplantable cells and tissues increases. Currently, several methods exist for the storage of cells and tissue. These methods include: (i) maintenance in tissue culture prior to transplantation, (ii) storage at refrigeration temperatures, and (iii) freeze drying. Most cells and tissues can be maintained in culture for only a few days, and are at risk for contamination by bacteria and other infectious agents. While refrigeration may prove useful for short term storage, most cells cannot be stored at refrigeration temperatures for prolonged periods or they begin to lose viability. Freeze drying is used for long term storage. However, many cells do not survive the freeze drying process.
Cryopreservation is one alternative to these methods. If not properly controlled, however, cryopreservation can lead to damage to the cells and a decrease in cell viability. Two major mechanisms of cell damage caused by cryopreservation have been reported. First, mechanical injuries to the cells are caused by formation of intracellular and extracellular ice crystal formation. See, e.g., U.S. Pat. No. 5,071,741. Damage caused by this mechanism is worse when cells are frozen at a rapid rate because rapid freezing causes an increase in the formation of intracellular and extracellular ice crystals. See, e.g., Karlsson et al., Biophysical J 65: 2524-2536, 1993. Penetrating cryopreservatives have been reported to alleviate damage caused by this first mechanism. See, e.g., U.S. Pat. No. 5,071,741, and Karlsson et al., Biophysical J 65: 2524-2536, 1993.
Second, cells may also be damaged by osmotic forces created by changing solute conditions caused by extracellular ice formation. As extracellular ice forms and continues to grow, water molecules become sequestrated within the ice, leaving solute molecules concentrated in the remaining fluidic fraction. This leads to a hyperosmotic extracellular environment. As a result of this osmotic imbalance, water transports out of the cell, causing the cells to shrink and leading to osmotic dehydration. Osmotic dehydration is exacerbated when cells are frozen at a slower rate, due to the tendency for ice crystals to form more rapidly in the extracellular medium under these conditions. Water ceases to cross the cell membrane when temperatures become sufficiently low to cause a phase transition in the cell's lipid bilayer, changing the lipid bilayer from a loosely packed alignment into a closely packed, semi-solid (gel) form with very limited permeability. See, e.g., PCT Publication No. WO 98/14058. Both penetrating and non-penetrating cryopreservatives have been reported to alleviate damage caused by this second mechanism. See, e.g., U.S. Pat. No. 5,071,741, PCT Publication No. WO 98/14058, Karlsson et al., Biophysical J 65: 2524-2536, 1993.
Optimal cryopreservation techniques must strike a balance between the damage caused to cells by mechanical forces during quick freezing and the damage caused to cells by osmotic forces during slow freezing. Different optimal cooling rates have been described for different cells. It has been suggested that the different optimal cooling rates are due to the differences in cellular ice nucleation constants and in phase transition temperature of the cell membrane for different cell types. See, e.g., PCT Publication No. WO 98/14058, and Karlsson et al., Biophysical J 65: 2524-2536, 1993. Freezing rates between -1.degree. C. per minute and -10.degree. C. per minute are preferred in the art. Karlsson et al., Biophysical J 65: 2524-2536, 1993.
Even when using cooling rates that are sufficiently fast to avoid damage by solution effects, intracellular ice formation will begin around -50.degree. C. Karlsson et al., Biophysical J 65: 2524-2536, 1993. When cells are frozen to temperatures below this level, intracellular ice nucleation is inevitable. Cells must be thawed quickly in order to avoid being extensively damaged during the thawing process by the expansive growth of these intracellular ice crystals. Warming cryopreserved cells to 37.degree. C. in 3 to 10 seconds will quickly bypass damage due to the crystallization phase and to any subsequent osmotic shock. Karlsson et al., Biophysical J 65: 2524-2536, 1993.
A polymer glass transition theory has been proposed to explain how cryoprotectants work. See, e.g., PCT Publication No. WO93/14191. The transition of an aqueous solution into an amorphous solid excludes formation of ice crystals. The glass transition temperature ("Tg") of a cell's aqueous environment is the temperature wherein a given solution goes from a glassy fluid to a rubbery state. Below the Tg, in the glass phase, the extremely high viscosities in the typical amorphous glass preclude molecular diffusion and hence chemical reactions that lead to cell damage. Two potential mechanisms are proposed. In the first mechanism, cryoprotectants work extracellularly by decreasing ice crystal formation and growth, thus reducing the movement of water molecules out of the cell during the freezing process. In the second mechanism, cryoprotectants work intracellularly by permeating the cell and reducing the amount of ice formed therein, hence reducing the amount of physical injury to cell membranes and organelles at transition phase temperatures.
Glycerol and dimethyl sulfoxide ("DMSO") have become the most widely accepted cryopreservative agents. Both of these compounds have very low Tg values, -65.degree. C. for a 40% (vol/vol) glycerol mixture and -120.degree. C. for a 5% (vol/vol) DMSO mixture. See, e.g., PCT Publication No. WO 93/14191. However, in order to be effective for the cryopreservation of cells or tissues encapsulated in BAOs, a cryoprotectant must not only maintain the integrity of the cellular membranes and the viability of the cells, but also maintain the integrity of the artificial jackets used to encapsulate the cells. This is critical to prevent either the release of the cellular contents into a patient upon transplantation, or allow components of a patient's immune system to permeate the protective membrane barrier. Moreover, the cryoprotectant mixture must be able to cross the semipermeable membrane in order to reach the encapsulated cells.
Although DMSO is a widely used cryopreservative of cells and tissues, it is unsuitable for use with BAOs. DMSO will dissolve the artificial polymer membrane surrounding the BAO, fracturing the integrity of the artificial jackets.
Glycerol cryopreservative compositions presently used in the art are also unsuitable for use with BAOs. Glycerol must be used in high concentrations for cryopreservation, namely, 40% (vol/vol) for red blood cells in combination with other cryoprotectants (e.g., PCT Publication No. WO 93/14191), up to 60-80% (weight to volume) for cultured skin equivalents and hematopoietic stem cells (e.g., PCT Publication Nos. WO 95/07611 and WO 96/27287, respectively). While concentrated glycerol solutions may be an effective cryoprotectant for cells in suspension, the limited wetting and diffusion properties of solutions of high concentrations of glycerol (e.g., PCT Publication No. WO 96/29862) does not permit adequate cryopreservation of cells or tissues in the microenvironment of BAOs.
Thus, a need remains in the art for viable methods and compositions for the cryopreservation of cells or tissues encapsulated in BAOs. Such methods and compositions would facilitate shipping and increase the shelf-life of BAOs.